Genes involved in the determination of segmental identity in Drosophila melanogaster have been identified on the basis of genetic interactions with homoeotic mutations, mutations already known to affect the process. Of the eighteen genes identified by interacting mutations, three act as negative regulators of homoeotic genes, and the remaining fifteen appear to be positive regulators, ancillary factors, or targets of homoeotic gene function. Of the latter fifteen, the genes kismet and Sex combs reduced behave as targets for the homoeotic gene Antennapedia. The genes brahma, osa, moira, skuld, kohtalo, and sallimus behave as ancillary factors for Antennapedia function, with at least brahma also acting as a positive regulator of Antennapedia gene function. Mutations associated with insertions of a Drosophila transposable element have been isolated for the genes kismet, brahma, and osa. The sequence of the putative protein product of the brahma locus has been determined. A 55 kilobase region of the genome including the brahma locus has been characterized by molecular and genetic methods. Three transcription units in addition to the brahma locus have been identified within this region of DNA. Sequence analyses of cDNAs from two of the three transcription units predict putative protein products with extensive sequence similarities to mammalian proteins thought to be involved in signal transduction. One putative protein product has similarities to the catalytic subunit of cAMP-dependent protein kinase, while the other putative protein product is similar to a GTP-binding protein termed ARF (ADP-ribosylation factor). Approximately thirty-five mutations in essential genes in this chromosome region have been isolated. These mutations identify at least three genes in addition to the brahma locus.